1. Field of the Invention
The present invention generally relates to the differentiation of embryonic stem cells in tissue culture into specific cell lineages, particularly cells of alveolar epithelial type II phenotype. The invention also relates to the therapeutic use of such differentiated cells in vivo.
2. Description of Related Art
The alveolar epithelium covers more than 99% of the internal surface area of the lung and is composed of two major cell types, the alveolar type I (ATI) cell and the alveolar type II (ATII) cell. ATI cells are large flat cells through which exchange of CO2/O2 takes place. They cover about 95% of the alveolar surface and comprise approximately 40% of the alveolar epithelium and 8% of the peripheral lung cells. In contrast, ATII cells are small, cuboidal cells, which cover about 5% of the alveolar surface and comprise 60% of the alveolar epithelium and 15% of the peripheral lung cells. They are characterized by the unique ability to synthesize and secrete surfactant protein C (SPC) and by the distinct morphological appearance of inclusion bodies, known as lamellar bodies. Important functions of ATII cells are: (i) to synthesize, store, and secrete surfactant, which reduces surface tension preventing collapse of the alveolus, (ii) to transportions from the alveolar fluid into the interstitium, thereby minimizing alveolar fluid and maximizing gas exchange, (iii) to serve as progenitor cells for alveolar type I cells, which is particularly important during re-epithelialization of the alveolus after lung injury, and (iv) to provide pulmonary host defense by synthesizing and secreting several complement proteins including C3 and C5 (1-3) as well as numerous cytokines and interleukins that modulate lymphocyte, macrophage and neutrophil functions (4). Severe pulmonary diseases can be caused by deficiencies or genetic mutations in proteins synthesized by AII cells that are important in maintaining normal lung homeostasis. For example, cystic fibrosis is caused by mutations in the transmembrane conductance receptor (CFTR) (5). CFTR is an important regulator of Cl− and liquid transport in the lung (6-9), and is functionally expressed by human ATII cells, strongly suggesting a critical role for CFTR in regulating ion and fluid transport in the lung alveolus (8). In addition, ATII cells synthesis and secrete the serine protease inhibitor, alpha-1 antitrypsin (α-1AT), which also plays a key role in alveolar homeostasis by regulating protease imbalance and adjusting fluid clearance (10, 11), the importance of which is supported by the association of α-1AT deficiency with the development of pulmonary emphysema (12).
Embryonic stem (ES) cells isolated from the inner cell mass of blastocyst-stage embryos are undifferentiated, pluripotent cells (13, 14), which can be induced to differentiate in vitro into a wide range of different cell types (15-23). The potential clinical use of ES cells to regenerate or repair damaged tissue has fueled a tremendous amount of research activity to develop methods that promote the differentiation of ES cells into specific cell lineages. Because of its numerous important functions, including its ability to proliferate and differentiate into the easily damaged ATI cell, human ES cell-derived ATII (hES-ATII) cells are promising as a source of cells that could be used therapeutically to treat distal lung injury as well as pulmonary genetic disorders. Recently published data demonstrated that ES cells can be differentiated into ATII cells via embryonic body (EB) formation (24, 25) or co-culture of EBs with pulmonary mesenchyme (26). However, these procedures were not efficient, generating only a very small percentage of ES cell-derived ATII cells (27). A mixed population of cell derivatives, as those generated in these reports, will not be suitable for transplantation into the lung. In addition, the pluripotent cells in the differentiating cultures carry a significant risk of producing teratomas after transplantation in vivo. Therefore, a major prerequisite for using ATII cells therapeutically is to achieve a pure population of hES-ATII cells. Selection strategies such as those employing protein markers or drug-resistance genes under the control of cell-specific promoters may be highly effective in producing a pure culture of ES cell-derived donor cells (28).